Exosomes released from breast cancer carcinomas stimulate cell movement

Abstract

For metastasis to occur cells must communicate with to their local environment to initiate growth and invasion. Exosomes have emerged as an important mediator of cell-to-cell signalling through the transfer of molecules such as mRNAs, microRNAs, and proteins between cells. Exosomes have been proposed to act as regulators of cancer progression. Here, we study the effect of exosomes on cell migration, an important step in metastasis. We performed cell migration assays, endocytosis assays, and exosome proteomic profiling on exosomes released from three breast cancer cell lines that model progressive stages of metastasis. Results from these experiments suggest: (1) exosomes promote cell migration and (2) the signal is stronger from exosomes isolated from cells with higher metastatic potentials; (3) exosomes are endocytosed at the same rate regardless of the cell type; (4) exosomes released from cells show differential enrichment of proteins with unique protein signatures of both identity and abundance. We conclude that breast cancer cells of increasing metastatic potential secrete exosomes with distinct protein signatures that proportionally increase cell movement and suggest that released exosomes could play an active role in metastasis.

Fig 1. Characterization of invasiveness associated with non-metastatic and metastatic breast cancer cell lines.

(A) Punctate expression pattern and cellular extensions of MCF-7 cells stably transfected with GFP-Rab27b-expressing plasmids. (B) Cells overexpression GFP-Rab27b formed cellular extensions with peripherally-localized GFP-Rab27b, and a spreading morphology on collagen-coated coverslips. (C) Measurement of cell proliferation of MCF-7 cells stably expression GFP or GFP-Rab27b. The same number of cells was plated in triplicate into 10 cm dishes on day 1 and the total number of cells was counted on day 8. (D) Matrigel invasion assay with MCF-7 GFP, MCF-7 Rab27b GFP, and MDA-MB-231cells. 10⁵ cells were seeded in serum free media on a Matrigel-coated filter and their migration toward medium containing serum was quantified by microscopic evaluation. MCF-7 cells stably overexpressing GFP-Rab27b showed significant increased invasion into matrigel compared to MCF-7 GFP cells (mean = 55.7 vs. 14.7 cells, P<0.0001). The well-characterized invasive MDA-MB-231 cell line showed massive invasion into matrigel, which has been described previously [62]. (E) The mean total number of invading cells from 10 different fields is shown P values were calculated using two-sided Student t tests. Statistically significant P values are indicated (n = 3); (F) Western blot analysis of protein extracts from MCF-7, MDA-MB-231, MCF-7 /GFP, MCF-7/GFP-RAB27a, and MCF-7/GFP-RAB27b probed with antibodies to GFP, RAB27a, and RAB27b. An anti-HRP conjugated β-actin antibody was used as a protein loading control. The established MCF-7 RAB27b GFP cell line showed over-expression of GFP-RAB27b demonstrated with an anti-GFP antibody, RAB27b antibody, and RAB27a antibody. Lysates of previously described cell lines (MCF-7 GFP, MCF-7 RAB27a GFP) were included as controls to show specificity of the antibodies [37].

Fig 2. Characterization of exosomes.

(A) Large-field view of electron micrograph of exosomes released from the human breast cancer cell line MCF-7 and stained with uranyl acetate. Scale bar, 100nm; (B) Magnified view of electron micrograph of exosomes released from the human breast cancer cell line MCF-7. Scale bars are 100nm. (C) Western blot analysis of exosomal proteins extracted from MCF-7, MCF-7/Rab27b, and MDA-MB-231.

Fig 3. Tumor-derived exosomes increase breast cell motility.

(A) Time course of cell migration wound-healing assays using MCF-7 breast cancer cells as the “recipient” cell line with the three “donor” exosome preparations. Serum-free media was used as a control. Each point on the assay represents three independent experiments at 21 hours. (B) Representative images from the wound healing experiments. Quantification (C) and images (D) of wound-healing assay for MCF7/Rab27b cells in the presence of the three exosome preparations and serum-free media after 19 hours. Quantification (C) and images (D) of wound-healing assay for MDA-MB-231 cells in the presence of the three exosome preparations and serum-free media after 15 hours. Errors were calculated from wound closure at each time point and normalized to the wound closure at the initial time point (0 hour). Experiments were repeated two additional times to verify results.

Fig 4. Endocytosis of exosomes in breast cancer cells.

(A) Time-course curve of exosome uptake (endocytosis) by determining fluorescent intensity of TAMRA-labeled exosomes from “donor” cancer cells at specific times. Inset shows a MCF-7 “recipient” cells incubated with TAMRA-labeled MCF-7 “donor” exosomes at 8 hours. Errors were calculated from fluorescence intensity of cells (n = 50), at each time point, normalized to the intensity at the final time point (24 hours). (B) Colocalization of TAMRA-labeled exosomes added to MCF-7 cells transiently transfected with LAMP1-GFP. Scale bar is 10μm.

Fig 5. Cytolocalization, molecular function, and the relative abundance of proteins identified in tumor-derived exosomes.

(A) Cellular localization of exosome proteins. Relative subcellular distribution of proteins identified in the exosomes isolated from the breast cancer cell line, MCF-7. Classification of the subcellular location of the proteins was based on the information provided by the UniProtKB/SwissProt database. (B) GO annotation of tumor-derived proteins. Proteins identified were allocated to different molecular function categories defined by the GO consortium. Relative protein levels of exosomes isolated from MCF-7 are indicated by the bars. Two independent biological replicates from the breast cancer cells type, MCF-7 were used for MS analysis. (C) iTRAQ analysis: Differential expression profile for metastatic tumor-derived exosome proteins in MDA-MB-231 and Rab27b-transformed MCF-7 cells compared to non-invasive breast cancer cells (MCF-7). Filled and open bars represent fold change in protein expression in Rab27b and MDA-MB-231 compared to MCF-7, respectively. Two independent biological replicates from the 3 cells types (MCF-7; Rab27b; MDA-MB-231) were used for iTRAQ analysis. The raw data give absolute expression levels of the various proteins (identity) and proteins levels (abundance) in all 3 cells lines. The average of proteins from the two independent samples from Rab27b and MDA-MB-231 lines was determined and compared to that the average of two samples from MCF-7 and the ratio for differential expression was determined.

Fig 6. Schematic representation of exosome composition and differentially expressed tumor-derived proteins.

(A) Cartoon of exosome with categories of identified associated proteins. (B) Table of differentially expressed proteins isolated from tumor-derived exosomes (MCF-7; Rab27b transformed MCF-7; MDA-MB-231) determined by iTRAQ analysis. The average ratio (≥2 fold) of differentially expressed exosomal proteins across MDA-MB-231/MCF-7 and Rab27b/MCF-7 were calculated and listed in the table.